In the prior art, to check the presence or absence of specific bacteria, such as foodborne pathogenic bacteria in foodstuff, bacteria are isolated from foodstuffs and cultured, and visual observation of the colonies (for color reactions and morphology of the colonies), microscopic observation, Gram-staining, biochemical testing and other tests are made. These methods require at least 2 days to demonstrate the presence or absence of the specific bacteria. Such methods can hardly be employed on manufacture s voluntary inspection of foodstuffs prior to shipment thereof since even though it can successfully detect bacteria, the resulting delay in taking due measures is intolerable.
In recent years, methods have been developed which comprise isolating bacteria from foodstuffs and culturing the same, and testing the thus-obtained colonies by the PCR method (Science, 230, 1350 (1985)) using primers corresponding to DNA of specific bacterial species to rapidly detect the specific bacterial DNA (Japanese Unexamined Patent Publications 1999-332600, 1995-236500, and 1993-317098). However, even when these methods are used, these methods require at least one day for isolation of bacteria from foodstuffs and cultivation of them.
DNAs coding for rRNAs (ribosomal ribonucleic acids) are often used as the targets for detection by the PCR method. This is because rRNAs exist in all organisms except for viruses and show high levels of homology among organisms of the same species because of relatively slow rates of evolution thereof. As regards bacterial 16S rRNA, in particular, numerous sequence data has been accumulated, therefore, DNAs encoding 16S rRNA seems to suit for detecting bacteria by the PCR method.
It has also been proposed that a direct PCR assay with broad-range PCR primers capable of hybridizing with many bacterial 16S rRNA-encoding DNAs are used for detecting a large variety of bacteria by use of bacterial DNA extracted from test samples (J. Dent. Res. 78(4): 850-856 (1999); J. Clinical Microbiology June 2000, 2076-2080; J. Clinical Microbiology February 1999, 464-466).
However, in cases where foodstuffs are used as test materials, the DNAs of the foodstuffs themselves are also extracted by the procedure for extracting bacterial DNAs. Therefore, if the primers are low in specificity, those chloroplast or mitochondrial DNAs derived from the foodstuffs, too, will be amplified, making the presence or absence of bacteria ambiguous.
To overcome such disadvantages, it is also possible to use primers capable of specifically hybridizing with the DNAs coding for the 16S rRNA of specific bacterial species (J. Applied Microbiology 1997, 83, 727-736). In this case, however, it becomes necessary to carry out tests separately for individual target bacterial species, hence the number of tests per foodstuff sample would increase. Thus, this method could hardly be employed by foodstuff manufacturers in voluntary testing.